The adrenal gland plays a crucial role in maintaining homeostasis during stressful situations by releasing cortical and medullary hormones into the blood stream. For over 10 years we have been particularly interested in the control of adrenal medulla by its preganglionic sympathetic neurons. These splanchnic neurons are cholinergic in nature and acetylcholine (ACh) released from the their terminals is the known stimulus of catecholamine secretion from chromaffin cells. In 1987 our analysis hinted that other substances, in addition to ACh could be released from nerve endings to stimulate the chromaffin cells. We used a series of physiological, pharmacological and immunohistochemical techniques together with radioimmunoassay to identify the nature of the non-cholinergic transmitter as vasoactive intestinal polypeptide (VIP). The new concept of non- cholinergic participation in the adrenal medulla has received wide acceptance because VIP, but not ACh, proved to be the stimulus for elevation of cAMP and consequent events in chromaffin cells. Within the last few years a new polypeptide, pituitary adenylate cyclase; activating polypeptide (PACAP), was identified as a member of the VIP-secretin family. PACAP has many similarities with VIP and has been found in neural tissues. These developments have opened new areas of research to further understand the physiological significance of multiple transmitter control of adrenal medullary secretion. In the present proposal we are raising three important questions. First, because of two lingering concerns regarding the role of VIP as a sole non-cholinergic transmitter, we question whether PACAP could be the other non-cholinergic transmitter. Second, our demonstration that ACh and VIP are released by different frequencies of splanchnic nerve stimulation raises the question of whether cholinergic and peptidergic transmitters are located in the same or different nerve endings. Third, because it is well known that there are two populations of chromaffin cells, epinephrine and norepinephrine rich, which lead to different proportions of the two catecholamines under different stressor conditions, we question whether ACh and peptides preferentially secrete epinephrine or norepinephrine. These issues will be addressed using techniques of immunohistochemistry, molecular biology, biochemistry and physiology with the combined expertise of our own laboratory and that of our most able colleagues.